Small molecules restore azole activity against drug-tolerant and drug-resistant Candida isolates.

Each year, fungi cause more than 1.5 billion infections worldwide and have a devastating impact on human health, particularly in immunocompromised individuals or patients in intensive care units. The limited antifungal arsenal and emerging multidrug-resistant species necessitate the development of new therapies. One strategy for combating drug-resistant pathogens is the administration of molecules that restore fungal susceptibility to approved drugs. Accordingly, we carried out a screen to identify small molecules that could restore the susceptibility of pathogenic Candida species to azole antifungals. This screening effort led to the discovery of novel 1,4-benzodiazepines that restore fluconazole susceptibility in resistant isolates of Candida albicans, as evidenced by 100-1,000-fold potentiation of fluconazole activity. This potentiation effect was also observed in azole-tolerant strains of C. albicans and in other pathogenic Candida species. The 1,4-benzodiazepines selectively potentiated different azoles, but not other approved antifungals. A remarkable feature of the potentiation was that the combination of the compounds with fluconazole was fungicidal, whereas fluconazole alone is fungistatic. Interestingly, the potentiators were not toxic to C. albicans in the absence of fluconazole, but inhibited virulence-associated filamentation of the fungus. We found that the combination of the potentiators and fluconazole significantly enhanced host survival in a Galleria mellonella model of systemic fungal infection. Taken together, these observations validate a strategy wherein small molecules can restore the activity of highly used anti-infectives that have lost potency. IMPORTANCE In the last decade, we have been witnessing a higher incidence of fungal infections, due to an expansion of the fungal species capable of causing disease (e.g., Candida auris), as well as increased antifungal drug resistance. Among human fungal pathogens, Candida species are a leading cause of invasive infections and are associated with high mortality rates. Infections by these pathogens are commonly treated with azole antifungals, yet the expansion of drug-resistant isolates has reduced their clinical utility. In this work, we describe the discovery and characterization of small molecules that potentiate fluconazole and restore the susceptibility of azole-resistant and azole-tolerant Candida isolates. Interestingly, the potentiating 1,4-benzodiazepines were not toxic to fungal cells but inhibited their virulence-associated filamentous growth. Furthermore, combinations of the potentiators and fluconazole decreased fungal burdens and enhanced host survival in a Galleria mellonella model of systemic fungal infections. Accordingly, we propose the use of novel antifungal potentiators as a powerful strategy for addressing the growing resistance of fungi to clinically approved drugs.

Thermoresponsive Copolymer Nanovectors Improve the Bioavailability of Retrograde Inhibitors in the Treatment of Leishmania Infections.

Clinical manifestations of leishmaniasis range from self-healing, cutaneous lesions to fatal infections of the viscera. With no preventative Leishmania vaccine available, the frontline option against leishmaniasis is chemotherapy. Unfortunately, currently available anti-Leishmania drugs face several obstacles, including toxicity that limits dosing and emergent drug resistant strains in endemic regions. It is, therefore, imperative that more effective drug formulations with decreased toxicity profiles are developed. Previous studies had shown that 2-(((5-Methyl-2-thienyl)methylene)amino)-N-phenylbenzamide (also called Retro-2) has efficacy against Leishmania infections. Structure-activity relationship (SAR) analogs of Retro-2, using the dihydroquinazolinone (DHQZ) base structure, were subsequently described that are more efficacious than Retro-2. However, considering the hydrophobic nature of these compounds that limits their solubility and uptake, the current studies were initiated to determine whether the solubility of Retro-2 and its SAR analogs could be enhanced through encapsulation in amphiphilic polymer nanoparticles. We evaluated encapsulation of these compounds in the amphiphilic, thermoresponsive oligo(ethylene glycol) methacrylate-co-pentafluorostyrene (PFG30) copolymer that forms nanoparticle aggregates upon heating past temperatures of 30°C. The hydrophobic tracer, coumarin 6, was used to evaluate uptake of a hydrophobic molecule into PFG30 aggregates. Mass spectrometry analysis showed considerably greater delivery of encapsulated DHQZ analogs into infected cells and more rapid shrinkage of L. amazonensis communal vacuoles. Moreover, encapsulation in PFG30 augmented the efficacy of Retro-2 and its SAR analogs to clear both L. amazonensis and L. donovani infections. These studies demonstrate that encapsulation of compounds in PFG30 is a viable approach to dramatically increase bioavailability and efficacy of anti-Leishmania compounds.

Discovery of Retro-1 Analogs Exhibiting Enhanced Anti-vaccinia Virus Activity.

Orthopoxviruses (OPXVs) are an increasing threat to human health due to the growing population of OPXV-naive individuals after the discontinuation of routine smallpox vaccination. Antiviral drugs that are effective as postexposure treatments against variola virus (the causative agent of smallpox) or other OPXVs are critical in the event of an OPXV outbreak or exposure. The only US Food and Drug Administration-approved drug to treat smallpox, Tecovirimat (ST-246), exerts its antiviral effect by inhibiting extracellular virus (EV) formation, thereby preventing cell-cell and long-distance spread. We and others have previously demonstrated that host Golgi-associated retrograde proteins play an important role in monkeypox virus (MPXV) and vaccinia virus (VACV) EV formation. Inhibition of the retrograde pathway by small molecules such as Retro-2 has been shown to decrease VACV infection in vitro and to a lesser extent in vivo. To identify more potent inhibitors of the retrograde pathway, we screened a large panel of compounds containing a benzodiazepine scaffold like that of Retro-1, against VACV infection. We found that a subset of these compounds displayed better anti-VACV activity, causing a reduction in EV particle formation and viral spread compared to Retro-1. PA104 emerged as the most potent analog, inhibiting 90% viral spread at 1.3 µM with a high selectivity index. In addition, PA104 strongly inhibited two distinct ST-246-resistant viruses, demonstrating its potential benefit for use in combination therapy with ST-246. These data and further characterizations of the specific protein targets and in vivo efficacy of PA104 may have important implications for the design of effective antivirals against OPXV.

Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins.

The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.